Tuberculosis (TB), a major cause of infectious disease deaths worldwide and particularly in developing regions, is a key global health challenge. The chronic disease is caused by M. tuberculosis and is transmitted primarily through mycobacterium-containing airborne droplet inhalation.
While TB has an 85% treatment success rate, the accurate M. tuberculosis detection challenge complicates TB control efforts. Timely treatment and diagnosis are important to control disease transmission due to TB’s serious effects and high infectivity.
Conventional TB diagnostic approaches, such as smear microscopy and culture, have several drawbacks. For instance, the long incubation period (2–6 weeks) of M. tuberculosis leads to delayed diagnosis. Similarly, smear microscopy has low sensitivity, particularly in patients with extrapulmonary TB or in samples with low bacterial loads.
Molecular diagnostic methods such as Next-Generation Sequencing (NGS) and Polymerase Chain Reaction (PCR) for TB diagnosis also have limitations, including expense, labor intensity, and time. Thus, developing new diagnostic approaches is crucial for affordable, sensitive, accurate, and fast disease detection.
NAA-based Biosensing Advances
Biosensors and nanotechnology have shown their potential for TB diagnosis. Among different nanomaterials, NAA is suitable for diagnostic tool development as it offers cost-efficient production through established techniques, surface modification ease, and a high loading capacity.
Additionally, NAA has recently been used for gated nanomaterial development for biosensing. Gated NAA-based biosensors are advantageous because they inherently amplify the signal, as a single analyte triggers the release of multiple encapsulated reporters. Previous research has demonstrated the effectiveness of gated NAA materials in detecting human papillomavirus and Staphylococcus aureus.
The Proposed Solution
In this work, researchers proposed a fluorogenic biosensor for M. tuberculosis MPT64 antigen detection using gated NAA. Here, NAA is loaded with a fluorescent reporter dye (rhodamine B) and capped with an antibody against the MPT64 protein (anti-MPT64), which specifically binds to MPT64.
Antibody displacement is induced by the presence of MPT64 due to specific recognition, and the subsequent release of rhodamine B into the medium is detected by fluorescence measurement. Researchers physicochemically characterized the biosensor using X-ray spectroscopy and scanning electron microscopy.
The use of antibodies can be a promising gating mechanism integrated into porous supports for functional sensing material development. Antibodies with high binding affinity for specific target molecules are well-suited for biosensors. Antibody-based biosensors have been used in medical diagnostics to detect biomarkers of various diseases, in biodefense, and for food safety.
In this work, researchers focused on the MPT64 protein as the target analyte, as it is a recognized biomarker of active TB and plays a key role in bacterial virulence. MPT64, as a secreted protein, enables antibody–analyte interaction without requiring bacterial lysis. This is a critical advantage of targeting MPT64.
MPT64 is also encoded in regions of difference and is thus absent in non-tuberculous mycobacteria and most Mycobacterium bovis bacille Calmette–Guérin (BCG) substrains, thereby improving detection specificity.
The antibody, acting as the gating mechanism, blocks the NAA pores and prevents dye release in the absence of the target protein. Conversely, the antibody binds to the protein in MPT64 presence, leading to pore opening and triggering dye release, generating a quantifiable fluorescence signal indicating M. tuberculosis presence. Thus, this method ensures rapid responses and high selectivity in TB diagnosis.
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Feasibility of the Proposed Biosensor
Researchers evaluated the sensitivity and selectivity of the M3 biosensor for detecting MPT64 protein. Experiments using varying concentrations of MPT64 displayed a direct relationship between protein concentration and dye release.
The limit of detection (LOD) was determined graphically by identifying the intersection between the fluorescence baseline and the positively sloped response curve, obtaining a value of 1.32 nM (0.032 mg L-¹). While this sensitivity was lower than that obtained from enzyme-linked immunosorbent assays, it was higher than that of electrochemical biosensors.
Selectivity tests demonstrated that the biosensor responded specifically to MPT64 as significant rhodamine B release occurred only in its presence, while other proteins associated with respiratory infections caused negligible responses, confirming high specificity. The strong performance was attributed to the synergistic effects of the NAA structure and its adaptable surface chemistry.
Robustness was further validated by testing in sputum fluid diluted in phosphate-buffered saline, where the biosensor maintained selective detection through antibody displacement and pore opening.
Clinical sample analysis showed higher fluorescence signals in samples from patients with M. tuberculosis, while samples with non-tuberculous mycobacteria produced signals comparable to negative controls, confirming reliable and selective detection.
In conclusion, the findings of this study demonstrated the great potential of the proposed biosensor as a clinical diagnostic tool for active TB.
Journal Reference
Caballos, I., Hernández-Montoto, A., Climent, E., Gil-Brusola, A., Martínez-Máñez, R., & Aznar, E. (2026). Targeted detection of Mycobacterium tuberculosis MPT64 antigen using an antibody-coated nanoporous anodic alumina biosensor: A novel approach for tuberculosis screening. Talanta, 305, 129625. DOI: 10.1016/j.talanta.2026.129625, https://www.sciencedirect.com/science/article/pii/S0039914026002808
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